Anti-plasma kallikrein antibodies

ABSTRACT

Disclosed herein are antibodies capable of binding to plasma kallikrein and inhibit its activity. Such antibodies interact with one or more critical residues in the catalytic domain of the plasma kallikrein. The antibodies may also contain specific heavy chain complementarity determining region 3 (CDRs) motifs and optionally specific residues at certain positions within both the heavy chain variable region and the light chain variable region.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/199,453, filed Nov. 26, 2018, which is a continuation of U.S.application Ser. No. 14/773,766, filed Sep. 9, 2015, which is a nationalstage filing under 35 U.S.C. § 371 of international PCT applicationPCT/US2014/027100, filed Mar. 14, 2014, which claims the benefit of U.S.Provisional Application No. 61/791,822, filed Mar. 15, 2013, each ofwhich is incorporated by reference herein in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference herein in its entirety. Said ASCII copy, created on Feb. 23,2022, is named D061770033US03-SEQ-EMB.txt and is 35,995 bytes in size.

BACKGROUND OF THE INVENTION

Plasma kallikrein is a serine protease component of the contact systemand a potential drug target for different inflammatory, cardiovascular,infectious (sepsis) and oncology diseases (Sainz I. M. et al., ThrombHaemost 98, 77-83, 2007). The contact system is activated by eitherfactor XIIa upon exposure to foreign or negatively charged surfaces oron endothelial cell surfaces by prolylcarboxypeptidases (Sainz I. M. etal., Thromb Haemost 98, 77-83, 2007). Activation of the plasmakallikrein amplifies intrinsic coagulation via its feedback activationof factor XII and enhances inflammation via the production of theproinflammatory nonapeptide bradykinin. As the primary kininogenase inthe circulation, plasma kallikrein is largely responsible for thegeneration of bradykinin in the vasculature. A genetic deficiency in theC1-inhibitor protein (C1-INH), the major natural inhibitor of plasmakallikrein, leads to hereditary angioedema (HAE). Patients with HAEsuffer from acute attacks of painful edema often precipitated by unknowntriggers (Zuraw B. L. et al., N Engl J Med 359, 1027-1036, 2008).

Through the use of pharmacological agents or genetic studies in animalmodels, the plasma kallikrein-kinin system (plasma KKS) has beenimplicated in various diseases. Thus, it is of great interest toidentify agents that inhibit plasma kallikrein activity, therebyeffective in treating diseases associated with plasma kallikrein.

SUMMARY OF THE INVENTION

The present invention is based on the determination of crystalstructures of a complex formed by the catalytic domain of human plasmakallikrein (PKal) and the Fab fragment of DX2930 (an antibodyspecifically binds human PKal and effectively inhibits its activity),and the identification of residues in both plasma kallirein (PKal) andthe antibody that are critical to the interaction between the twomolecules and/or to the inhibition of the pKal activity.

Accordingly, the present disclosure features anti-PKal antibodiescapable of inhibiting its activity (e.g., by at least 50%),pharmaceutical compositions comprising such, and uses of thepharmaceutical compositions for treating diseases and disordersassociated with plasma kallikrein.

In one aspect, the present disclosure provides an isolated antibody thatbinds human plasma kallikrein (PKal), wherein the antibody interactswith one or more of amino acid residues in the human PKal and inhibitsits activity by at least 50%. The amino acid residues in the PKal thatinteract with the antibody can be V410, L412, T413, A414, Q415, R416,L418, C419, H434, C435, F436, D437, G438, L439, W445, Y475, K476, V477,5478, E479, G480, D483, F524, E527, K528, Y552, D554, Y555, A564, D572,A573, C574, K575, G576, 5578, T596, 5597, W598, G599, E600, G601, C602,A603, R604, Q607, P608, G609, V610, and Y611 as indicated in FIG. 2(boldfaced and underlined).

In some examples, the anti-PKal antibody can bind an epitope of thePKal, the epitope comprising one of the following segments in PKal (FIG.2 ): V410-C419, H434-L439, Y475-G480, F524-K528, Y552-Y555, D572-S578,T596-R604, or Q607-Y611.

In other examples, the antibody preferentially binds the PKal asrelative to a mutant of the PKal (e.g., an inactive mutant) thatcontains one or more mutations at positions R551, Q553, Y555, T558, andR560 (e.g., Mutant 2 shown in FIG. 5 ).

In another aspect, the present disclosure provides an isolated antibodythat binds human plasma kallikrein, wherein the antibody comprises aheavy chain variable region that comprises complementarity determiningregion 1 (HC CDR1), complementarity determining region 2 (HC CDR2), andcomplementarity determining region 3 (HC CDR3). The HC CDR3 in theantibody comprises the motifX₉₉R₁₀₀X₁₀₁G₁₀₂X₁₀₃P₁₀₄R₁₀₅X₁₀₆X₁₀₇X₁₀₈X₁₀₉X₁₁₀X₁₁₁ (SEQ ID NO: 58), inwhich X₉₉ is R or Q, X₁₀₁ is T, I, R, S, or P, X₁₀₃ is V, I, or L, X₁₀₆is R or W, X₁₀₇ is D or N, X₁₀₈ is A, S, D, E, or V, X₁₀₉ is F or L,X₁₁₀ is D, E, or N, and X₁₁₁ is I, N, M, or S.

In some examples, X₉₉ can be Q and X₁₀₁ can be I, R, S, or P. In otherexamples, X₁₀₆ can be W and X₁₁₁ can be N, M, or S. Alternatively or inaddition, X₁₀₁ can be I, X₁₀₈ can be E, and X₁₀₃ can be I or L. In yetother examples, X₁₀₁ can be I and X₁₀₃ can be I or L, or X₁₀₃ can be Ior L and X₁₁₀ can be D, E, or N.

In some embodiments, the heavy chain variable region of the anti-PKalantibody described herein includes H₃₁ in the HC CDR1. Alternatively orin addition, the heavy chain variable region includes F₂₇, F₂₉, or bothin the framework region 1 (FR1).

The anti-PKal antibody described herein can further comprise a lightchain variable region that comprises complementarity determining region1 (LC CDR1), complementarity determining region 2 (LC CDR2), andcomplementarity determining region 3 (LC CDR3). In some embodiments, theLC CDR2 includes K₅₀, L₅₄, E₅₅, S₅₆, or a combination thereof.Alternatively or in addition, the light chain variable region furtherincludes G₅₇ in the framework region 3 (FR3). When necessary, the lightchain variable includes N₄₅ in the framework region 2 (FR2).

Any of the anti-PKal antibodies described herein can inhibit theactivity of PKal by at least 50% (e.g., at least 80%, 90%, 95%, or 99%).In some instances, the antibody has an apparent Ki (K_(i,app)) lowerthan about 1 nM (e.g., lower than about 0.1 nM, or lower than about 0.05nM). Alternatively or in addition, the anti-PKal antibody describedherein can have a binding affinity (K_(D)) for the PKal of less than10⁻⁶ M (e.g., less than 10⁻⁷ M, 10⁻⁸ M, or 10⁻⁹ M).

The anti-PKal antibodies described herein can be a full-length antibodyor an antigen-binding fragment thereof. Alternatively or in addition,the antibody can be a human antibody or a humanized antibody.

Also within the scope of the present disclosure are pharmaceuticalcompositions for use in treating various diseases and disordersassociated with plasma kallikrein, or for use in manufacturing amedicament for treating the diseases and disorders. The pharmaceuticalcompositions each comprise one or more anti-PKal antibodies as describedherein and a pharmaceutically acceptable carrier.

Further, described herein are methods for treating a disease associatedwith plasma kallikrein, comprising administering to a subject in needthereof an effective amount of the pharmaceutical composition, whichcomprises one or more of the anti-PKal antibodies described herein. Insome examples, the subject is a human patient diagnosed with, suspectedof having, or at risk for the disease.

The details of one or more embodiments of the invention are set forth inthe description below. Other features or advantages of the presentinvention will be apparent from the following drawings and detaileddescription of several embodiments, and also from the appending claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentdisclosure, which can be better understood by reference to one or moreof these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1 shows the amino acid sequence of the heavy chain variable region(V_(H)) and light chain variable region (V_(L)) of a parent antibody,M0162-A04, from which DX2930 was derived, and their alignment with thecorresponding germline V_(H) and V_(L) genes as indicated. The M0162-A04V_(L) complementarity determining region (CDR) 1 corresponds to SEQ IDNO: 59, V_(L) CDR2 corresponds to SEQ ID NO: 60, V_(L) CDR3 correspondsto SEQ ID NO: 61, V_(H) CDR1 corresponds to SEQ ID NO: 62. V_(H) CDR2corresponds to SEQ ID NO: 63, and V_(H) corresponds to SEQ ID NO: 64.Variations in M0162-A04 as compared to the germline sequences areindicated (boldfaced).

FIG. 2 shows the amino acid sequence (SEQ ID NO:40) of the catalyticdomain of human plasma kallikrein (residues 391-638 of the full lengthhuman PKal). The boldfaced and underlined residues refer to those thatare involved in the interaction with the Fab fragment of DX2930 asidentified by the crystal structure discussed in Example 1 below.

FIG. 3 is a graph showing the apparent Ki (K_(i,app)) of a number ofantibody mutants derived from M0162-A04 against human PKal.

FIG. 4 is a graph showing the apparent Ki (K_(i,app)) of clone X115-F02(see Table 1 below) against wild-type PKal and a number of PKal mutants.

FIG. 5 shows the amino acid sequences of a number of PKal mutants(catalytic domain), which were produced in Pichia cells.

DETAILED DESCRIPTION OF THE INVENTION

DX-2930 is a fully human IgG derived from parent clone M0162-A04. Theamino acid sequences of the V_(H) and V_(L) of M162-A04 are shown inFIG. 1 . Their alignment with the corresponding germline VH gene(VH3_3-23) and VL gene (VK1_L12) is also shown in FIG. 1 . Compared tothe HC CDR3 of M0162-A04, the HC CDR3 of DX-2930 includes the variationsof T101I, I103V, and A108E (see Table 2 below; the HC CDR3 of DX-2930being identical to M0199-A08). The Chothia Numbering Scheme is used inthe present disclosure. www.bioinf.org.uk/abs/.

Table 1 below provides structural information of DX-2930, its parentantibody M0162-A04, and variants thereof. See also US20120201756 andUS20110200611.

TABLE 1 Structural Properties of DX-2930 and Related Variants NameProperties M162-A04 This is the parent antibody of DX-2930 that wasdiscovered in the initial phage display selection efforts (Ki, app = 2.5nM). This antibody differs from DX-2930 at 3 critical amino acids in theCDR3 of the heavy chain and the germlined positions. M199-A08 Fabdiscovered following the affinity maturation of M0162-A04 using theHv-CDR3 spiking method (Ki, app~0.06 nM). This antibody shares the sameamino acids in the variable region with DX-2930 but was not germlinedand does not contain a Fc fragment. X115-F02 Fully human IgG, kappalight chain 1 amino acid in the light chain was mutated to theirgermline sequence. The DNA sequence of X115-F02 was optimized forexpression in CHO cells Expressed transiently in 293T cells followingsubcloning into the pRH1-CHO vector DX-2930 Fully human IgG, kappa lightchain (X124-G01) 1 amino acid in the light chain and 2 amino acids inthe heavy were mutated to their germline sequence. The DNA sequence ofDX-2930 was optimized for expression in CHO cells and cloned into thepEh1 vector for stable expression using the glutamate synthase system.The Fc of DX-2930 was modified to remove the C- terminal lysine reside,in order to obtain a more homogeneous product.

Crystal structures (with different resolutions) of a complex formedbetween the Fab fragment of DX-2930 and the catalytic domain of humanplasma kallikrein (PKal) was determined. Based on the structuralinformation provided by the crystal structures, a number of interactingresidues in both the catalytic domain of human PKal and the antibody (inboth V_(H) and V_(L)) were identified. The interacting residues in thePKal are important targets for developing antibodies capable ofinhibiting the PKal activity. Similarly, the interacting residues in theantibody also provide important structural information for designinganti-PKal antibodies with high inhibitory activity.

Further, affinity maturation analysis was performed to develop highaffinity anti-PKal antibodies, using clone M0162-A04 as the parent.Results obtained from affinity maturation matches with the structuralinformation provided by the crystal structures. Based on the structuralinformation and the affinity maturation results, specific VH and VLmotifs/residues were identified for designing anti-PKal antibodies withhigh inhibitory activities.

Accordingly, described herein are antibodies capable of binding toplasma kallikrein (e.g., human plasma kallikrein; PKal) and inhibitingits activity, and uses thereof for treating diseases and disordersassociated with plasma kallikrein. Such antibodies interact with one ormore critical residues in the catalytic domain of the PKal and/orcomprise specific motifs/residues in either the heavy chain variableregion (e.g., HC CDR1 or HC CDR3) or the light chain variable region(e.g., LC CDR2), or both.

Antibodies Binding to PKal

The present disclosure provides isolated antibodies that bind PKal,particularly the catalytic domain of the PKal, such as human PKal. Theterm “isolated antibody” used herein refers to an antibody substantiallyfree from naturally associated molecules, i.e., the naturally associatedmolecules constituting at most 20% by dry weight of a preparationcontaining the antibody. Purity can be measured by any appropriatemethod, e.g., column chromatography, polyacrylamide gel electrophoresis,and HPLC.

An antibody (interchangeably used in plural form) is an immunoglobulinmolecule capable of specific binding to a target, such as acarbohydrate, polynucleotide, lipid, polypeptide, etc., through at leastone antigen recognition site, located in the variable region of theimmunoglobulin molecule. As used herein, the term “antibody” encompassesnot only intact (i.e., full-length) polyclonal or monoclonal antibodies,but also antigen-binding fragments thereof (such as Fab, Fab′, F(ab′)2,Fv), single chain (scFv), mutants thereof, fusion proteins comprising anantibody portion, humanized antibodies, chimeric antibodies, diabodies,linear antibodies, single chain antibodies, multispecific antibodies(e.g., bispecific antibodies) and any other modified configuration ofthe immunoglobulin molecule that comprises an antigen recognition siteof the required specificity, including glycosylation variants ofantibodies, amino acid sequence variants of antibodies, and covalentlymodified antibodies. An antibody includes an antibody of any class, suchas IgD, IgE, IgG, IgA, or IgM (or sub-class thereof), and the antibodyneed not be of any particular class. Depending on the antibody aminoacid sequence of the constant domain of its heavy chains,immunoglobulins can be assigned to different classes. There are fivemajor classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, andseveral of these may be further divided into subclasses (isotypes),e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy-chain constantdomains that correspond to the different classes of immunoglobulins arecalled alpha, delta, epsilon, gamma, and mu, respectively. The subunitstructures and three-dimensional configurations of different classes ofimmunoglobulins are well known.

The antibodies described herein are capable of binding to a PKal,particularly the catalytic domain of a PKal (e.g., human PKal), therebyinhibiting the activity of PKal. In some instances, the antibodiesdescribed herein can inhibit the activity of PKal by at least 50%, e.g.,60%, 70%, 80%, 90%, 95%, or higher. The inhibition constant (Ki)provides a measure of inhibitor potency; it is the concentration ofinhibitor required to reduce enzyme activity by half and is notdependent on enzyme or substrate concentrations. The inhibitory activityof an anti-PKal antibody can be determined by routine methods, such asthe method described in Example 2 below.

In some examples, the inhibitory activity of an anti-PKal antibody isdetermined by the apparent Ki (K_(i,app)) value. The K_(i,app) value ofan antibody obtained at different substrate concentrations by measuringthe inhibitory effect of different concentrations of the antibody on theextent of the reaction (e.g., enzyme activity); fitting the change inpseudo-first order rate constant as a function of inhibitorconcentration to the Morrison equation (Equation 1) yields an estimateof the apparent Ki value. For a competitive inhibitor, the Ki isobtained from the y-intercept extracted from a linear regressionanalysis of a plot of K_(i,app) versus substrate concentration.

$\begin{matrix}{v = {v_{o} - {v_{o}\left( \frac{\left( {K_{i,{app}} + I + E} \right) - \sqrt{\left( {K_{i,{app}} + I + E} \right)^{2} - {4 \cdot I \cdot E}}}{2 \cdot E} \right)}}} & {{Equation}1}\end{matrix}$

In some examples, the anti-PKal antibodies described herein have aK_(i,app) value lower than 1 nM, e.g., 0.5 nM, 0.2 nM, 0.1 nM, 0.09 nM,0.08 nM, 0.07 nM, 0.06 nM, 0.05 nM, 0.04 nM, 0.03 nM, 0.02 nM, 0.01 nM,or lower. The K_(i,app) value of an antibody can be estimated followingthe methods known in the art and described herein (Example 2).

The antibodies described herein can be murine, rat, human, or any otherorigin (including chimeric or humanized antibodies). In some examples,the antibody comprises a modified constant region, such as a constantregion that is immunologically inert, e.g., does not trigger complementmediated lysis, or does not stimulate antibody-dependent cell mediatedcytotoxicity (ADCC). ADCC activity can be assessed using methodsdisclosed in U.S. Pat. No. 5,500,362. In other embodiments, the constantregion is modified as described in Eur. J. Immunol. (1999) 29:2613-2624;PCT Application No. PCT/GB99/01441; and/or UK Patent Application No.9809951.8.

Any of the antibodies described herein can be either monoclonal orpolyclonal. A “monoclonal antibody” refers to a homogenous antibodypopulation and a “polyclonal antibody” refers to a heterogeneousantibody population. These two terms do not limit the source of anantibody or the manner in which it is made.

In one example, the antibody used in the methods described herein is ahumanized antibody. Humanized antibodies refer to forms of non-human(e.g. murine) antibodies that are specific chimeric immunoglobulins,immunoglobulin chains, or antigen-binding fragments thereof that containminimal sequence derived from non-human immunoglobulin. For the mostpart, humanized antibodies are human immunoglobulins (recipientantibody) in which residues from a complementary determining region(CDR) of the recipient are replaced by residues from a CDR of anon-human species (donor antibody) such as mouse, rat, or rabbit havingthe desired specificity, affinity, and capacity. In some instances, Fvframework region (FR) residues of the human immunoglobulin are replacedby corresponding non-human residues. Furthermore, the humanized antibodymay comprise residues that are found neither in the recipient antibodynor in the imported CDR or framework sequences, but are included tofurther refine and optimize antibody performance. In general, thehumanized antibody will comprise substantially all of at least one, andtypically two, variable domains, in which all or substantially all ofthe CDR regions correspond to those of a non-human immunoglobulin andall or substantially all of the FR regions are those of a humanimmunoglobulin consensus sequence. The humanized antibody optimally alsowill comprise at least a portion of an immunoglobulin constant region ordomain (Fc), typically that of a human immunoglobulin. Antibodies mayhave Fc regions modified as described in WO 99/58572. Other forms ofhumanized antibodies have one or more CDRs (one, two, three, four, five,six) which are altered with respect to the original antibody, which arealso termed one or more CDRs “derived from” one or more CDRs from theoriginal antibody.

Humanized antibodies may also involve affinity maturation.

In another example, the antibody described herein is a chimericantibody, which can include a heavy constant region and a light constantregion from a human antibody. Chimeric antibodies refer to antibodieshaving a variable region or part of variable region from a first speciesand a constant region from a second species. Typically, in thesechimeric antibodies, the variable region of both light and heavy chainsmimics the variable regions of antibodies derived from one species ofmammals (e.g., a non-human mammal such as mouse, rabbit, and rat), whilethe constant portions are homologous to the sequences in antibodiesderived from another mammal such as human. In some embodiments, aminoacid modifications can be made in the variable region and/or theconstant region.

In some embodiments, the anti-PKal antibodies described herein have asuitable binding affinity to a PKal or the catalytic domain thereof. Asused herein, “binding affinity” refers to the apparent associationconstant or K_(A). The K_(A) is the reciprocal of the dissociationconstant (K_(D)). The antibody described herein may have a bindingaffinity (K_(D)) of at least 10⁻⁵, 10-6, 10⁻⁷, 10-8, 10-9, 10⁻¹⁰ M, orlower. An increased binding affinity corresponds to a decreased K_(D).Higher affinity binding of an antibody to a first target relative to asecond target can be indicated by a higher K_(A) (or a smaller numericalvalue K_(D)) for binding the first target than the K_(A) (or numericalvalue K_(D)) for binding the second target. In such cases, the antibodyhas specificity for the first target (e.g., a protein in a firstconformation or mimic thereof) relative to the second target (e.g., thesame protein in a second conformation or mimic thereof; or a secondprotein). Differences in binding affinity (e.g., for specificity orother comparisons) can be at least 1.5, 2, 3, 4, 5, 10, 15, 20, 37.5,50, 70, 80, 91, 100, 500, 1000, 10,000 or 10⁵ fold.

Binding affinity can be determined by a variety of methods includingequilibrium dialysis, equilibrium binding, gel filtration, ELISA,surface plasmon resonance, or spectroscopy (e.g., using a fluorescenceassay). Exemplary conditions for evaluating binding affinity are inHBS-P buffer (10 mM HEPES pH7.4, 150 mM NaCl, 0.005% (v/v) SurfactantP20). These techniques can be used to measure the concentration of boundbinding protein as a function of target protein concentration. Theconcentration of bound binding protein ([Bound]) is related to theconcentration of free target protein ([Free]) and the concentration ofbinding sites for the binding protein on the target where (N) is thenumber of binding sites per target molecule by the following equation:

[Bound]=[N][Free]/(Kd+[Free])

It is not always necessary to make an exact determination of K_(A),though, since sometimes it is sufficient to obtain a quantitativemeasurement of affinity, e.g., determined using a method such as ELISAor FACS analysis, is proportional to K_(A), and thus can be used forcomparisons, such as determining whether a higher affinity is, e.g.,2-fold higher, to obtain a qualitative measurement of affinity, or toobtain an inference of affinity, e.g., by activity in a functionalassay, e.g., an in vitro or in vivo assay.

Antibodies Targeting Specific Residues in Human Plasma Kallikrein

In some embodiments, the anti-PKal antibodies interact with one or moreof the residues (e.g., at least 3, 5, 8, 10, 15, 20, 25, 30, 35, 40, or45) in the catalytic domain of human PKal, including V410, L412, T413,A414, Q415, R416, L418, C419, H434, C435, F436, D437, G438, L439, W445,Y475, K476, V477, S478, E479, G480, D483, F524, E527, K528, Y552, D554,Y555, A564, D572, A573, C574, K575, G576, S578, T596, S597, W598, G599,E600, G601, C602, A603, R604, Q607, P608, G609, V610, and Y611 (numbersbased on the full length prekallikrein amino acid sequence). Thepositions of these residues are indicated in FIG. 2 (boldfaced andunderlined). These residues are identified as important to the pKalactivity, according to the crystal structures described in Example 1below.

In some embodiments, the anti-PKal antibodies interact with one or moreof the residues (e.g., at least 3, 5, 8, 10, 15, 20, or 23) in thecatalytic domain of human PKal, including L418, C419, H434, C435, D437,G438, L439, Y475, D483, F524, D572, A573, C574, K575, G576, S578, T596,S597, W598, G599, E600, G601, and C602 (numbers based on the full lengthprekallikrein amino acid sequence).

In some embodiments, the anti-PKal antibodies interact with one or moreof the residues (e.g., at least 3, 5, or 8) in the catalytic domain ofhuman PKal, including K476, V477, S478, E479, G480, Y552, D554, and Y555(numbers based on the full length prekallikrein amino acid sequence).

In some embodiments, the anti-PKal antibodies interact with one or moreof the residues (e.g., at least 3, 5, 8, or 10) in the catalytic domainof human PKal, including V410, L412, T413, A414, Q415, R416, E527, K528,A603, and R604 (numbers based on the full length prekallikrein aminoacid sequence).

In some embodiments, the anti-PKal antibodies interact with one or moreof the residues (e.g., at least 3, 5, or 6) in the catalytic domain ofhuman PKal, including W445, Q607, P608, G609, V610, and Y611 (numbersbased on the full length prekallikrein amino acid sequence).

In some embodiments, the anti-PKal antibodies interact with one or moreof the residues (e.g., at least 3, 5, 8, or 9) in the catalytic domainof human PKal, including F524, D572, A573, C574, K575, G576, S578, G601,and C602 (numbers based on the full length prekallikrein amino acidsequence).

In some embodiments, the anti-PKal antibodies interact with one or moreof the residues (e.g., at least 3, 5, or 8) in the catalytic domain ofhuman PKal, including L418, C419, H434, C435, D437, G438, Y475, and D483(numbers based on the full length prekallikrein amino acid sequence).

In some embodiments, the anti-PKal antibodies interact with one or moreof the residues (e.g., at least 3 or 4) in the catalytic domain of humanPKal, including S597, W598, G599, and E600 (numbers based on the fulllength prekallikrein amino acid sequence).

Interacting means that the distance between two residues in a complexformed by two binding partners is lower than a predetermined value,e.g., <6 Å, <4 Å, or <2 Å. For example, an interacting residue in onebinding partner can have has at least 1 atom within a given threshold(e.g., <6 Å, <4 Å, or <2 Å) of at least 1 atom from a residue of theother binding partner on the complexed structure. Interacting does notrequire actual binding. Interacting residues are suggested as involvedin antibody recognition.

In some embodiments, the antibodies described herein bind human PKal atan epitope comprising one or more of the residues listed above. An“epitope” refers to the site on a target compound that is bound by anantibody such as a Fab or full length antibody. An epitope can belinear, which is typically 6-15 aa in length. Alternatively, the epitopecan be conformational.

In some examples, the anti-PKal antibodies described herein binds anepitope that comprises the following segments: V410-C419, H434-L439,Y475-G480, F524-K528, Y552-Y555, D572-S578, T596-R604, or Q607-Y611.

In some examples, the antibody disclosed herein specifically binds PKalor an epitope therein. An antibody that “specifically binds” (usedinterchangeably herein) to a target or an epitope is a term wellunderstood in the art, and methods to determine such specific bindingare also well known in the art. A molecule is said to exhibit “specificbinding” if it reacts or associates more frequently, more rapidly, withgreater duration and/or with greater affinity with a particular targetantigen than it does with alternative targets. An antibody “specificallybinds” to a target antigen if it binds with greater affinity, avidity,more readily, and/or with greater duration than it binds to othersubstances. For example, an antibody that specifically (orpreferentially) binds to human PKal or an epitope therein is an antibodythat binds this target antigen with greater affinity, avidity, morereadily, and/or with greater duration than it binds to other antigens orother epitopes in the same antigen. It is also understood by readingthis definition that, for example, an antibody that specifically bindsto a first target antigen may or may not specifically or preferentiallybind to a second target antigen. As such, “specific binding” or“preferential binding” does not necessarily require (although it caninclude) exclusive binding. Generally, but not necessarily, reference tobinding means preferential binding.

In one example, the anti-PKal antibodies described herein preferentiallybind wild-type as compared to a mutant that includes mutations at one ormore of R551, Q553, Y555, T558, and R560, e.g., Mutant 2 described inExample 3. Such antibodies may bind wild-type PKal at a much higheraffinity as compared to the mutant (e.g., at least 2-fold, 5-fold,10-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1,000-fold higher).Alternatively or in addition, the antibodies exhibit a much higherinhibitory activity against the wild-type pKal as relative to the mutant(e.g., at least 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 200-fold,500-fold, 1,000-fold higher).

In other examples, the anti-PKal antibodies described herein bindsactive PKal, including wild-type pKal and functional variant thereof.The antibody can preferentially bind an active PKal as relative to itsbinding to an inactive mutant.

Anti-Plasma Kallikrein Antibodies Having Specific Motifs and/or Residues

In some embodiments, the anti-PKal antibody described herein comprises aV_(H) and a V_(L), each of which comprises three CDRs flanked byframework regions (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4; see FIG. 1 ). TheCDR3 of the heavy chain can comprise the motif:X₉₉R₁₀₀X₁₀₁G₁₀₂X₁₀₃P₁₀₄R₁₀₅X₁₀₆X₁₀₇X₁₀₈X₁₀₉X₁₁₀X₁₁₁, in which X₉₉ is Ror Q, X₁₀₁ is T, I, R, S, or P, X₁₀₃ is V, I, or L, X₁₀₆ is R or W, X₁₀₇is D or N, X₁₀₈ is A, S, D, E, or V, X₁₀₉ is F or L, X₁₁₀ is D, E, or N,and X₁₁₁ is I, N, M, or S. In some examples, X₉₉ is Q and X₁₀₁ is I, R,S, or P. Alternatively or in addition, X₁₀₆ is W and X₁₁₁ is N, M, or S.In other examples, X₁₀₁ is I, X₁₀₈ is E, and X₁₀₃ is I or L; or X₁₀₁ isI and X₁₀₃ is I or L. In yet other examples, X₁₀₃ is I or L and X₁₁₀ isD, E, or N.

In addition, such an anti-pKal antibody can include one or more otherresidues that are identified based on the crystal structures discussedherein as being involved in interacting with the catalytic domain ofhuman PKal. These residues can be located in the V_(H) or the V_(L)chain. Examples include E1, V2, F27, T28, F29, and S30 in the FR1 of theV_(H), H₃₁ in the HC CDR1; S31 and W32 in the LC CDR1, Y49 in the FR1 ofthe V_(L) chain, K50, T53, L54, and E55, and S56 in LC CDR2, and G57 andV58 the FR3 of the V_(L) chain.

The anti-PKal antibodies as described above can use any germline heavychain and light chain V genes as the framework. Heavy chain V genesinclude, but are not limited to, IGHV1-2, IGHV1-3, IGHV1-8, IGHV1-18,IGHV1-24, IGHV1-45, IGHV1-46, IGHV1-58, IGHV1-69, IGHV2-5, IGHV2-26,IGHV2-70, IGHV3-7, IGHV3-9, IGHV3-11, IGHV3-13, IGHV3-15, IGHV3-20,IGHV3-21, IGHV3-23, IGHV3-30, IGHV3-33, IGHV3-43, IGHV3-48, IGHV3-49,IGHV3-53, IGHV3-64, IGHV3-66, IGHV3-72, IGHV3-73, IGHV3-74, IGHV4-4,IGHV4-28, IGHV4-31, IGHV4-34, IGHV4-39, IGHV4-59, IGHV4-61, IGHV4-B,IGHV5-51, IGHV6-1, and IGHV7-4-1.

In some examples, the antibody uses a κ light chain. Light chain VKgenes include, but are not limited to, V genes for IGKV1-05, IGKV1-06,IGKV1-08, IGKV1-09, IGKV1-12, IGKV1-13, IGKV1-16, IGKV1-17, IGKV1-27,IGKV1-33, IGKV1-37, IGKV1-39, IGKV1D-16, IGKV1D-17, IGKV1D-43, IGKV1D-8,IGKV2-24, IGKV2-28, IGKV2-29, IGKV2-30, IGKV2-40, IGKV2D-26, IGKV2D-29,IGKV2D-30, IGKV3-11, IGKV3-15, IGKV3-20, IGKV3D-07, IGKV3D-11,IGKV3D-20, IGKV4-1, IGKV5-2, IGKV6-21, and IGKV6D-41. In other examples,the antibody uses a λ light chain, e.g., any of IGLV1-IGLV10.

The antibody also can use any germline heavy J segment (e.g., heavychain IGJH1-IGJH6) and light chain J segment (e.g., IGJK1, IGJK2, IGJK3,IGJK4, or IGJK5), which can subject to variations, such as deletions atthe C-terminus, N-terminus, or both.

Germline antibody gene/segment sequences are well known in the art. See,e.g., www.vbase2.org/vbstat.php.

In some examples, the anti-PKal antibody described herein uses VH3_3-23and/or VK1_L12 as the framework for the heavy chain and/or the lightchain. It may include substantially similar HC CDR1, HC CDR2, and/or HCCDR3, and LC CDR1, LC CDR2, and/or LC CDR3 as those in M0162-A04 (FIG. 1), e.g., containing up to 5, 4, 3, 2, or 1 amino acid residue variationsas compared to the corresponding CDR region in M0162-A04.

In other examples, the anti-PKal antibody comprises a V_(H) chain thatincludes a V_(H) CDR1, V_(H) CDR2, and V_(H) CDR3 at least 75% (e.g.,80%, 85%, 90%, 95%, or 98%) identical to the corresponding V_(H) CDRs ofM0162-A04, and a V_(L) chain that includes a V_(L) CDR1, V_(L) CDR2, andV_(L) CDR3 at least 75% (e.g., 80%, 85%, 90%, 95%, or 98%) identical tothe corresponding V_(L) CDRs of M0162-A04.

Alternatively, the anti-PKal antibody comprises a V_(H) chain at least75% (e.g., 80%, 85%, 90%, 95%, or 98%) identical to the V_(H) chain(mature or precursor) of M0162-A04 and/or a V_(L) chain at least 75%(e.g., 80%, 85%, 90%, 95%, or 98%) identical to the V_(L) chain (matureof precursor) of M0162-A04.

The “percent identity” of two amino acid sequences is determined usingthe algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA87:2264-68, 1990, modified as in Karlin and Altschul Proc. Natl. Acad.Sci. USA 90:5873-77, 1993. Such an algorithm is incorporated into theNBLAST and XBLAST programs (version 2.0) of Altschul, et al. J. Mol.Biol. 215:403-10, 1990. BLAST protein searches can be performed with theXBLAST program, score=50, wordlength=3 to obtain amino acid sequenceshomologous to the protein molecules of interest. Where gaps existbetween two sequences, Gapped BLAST can be utilized as described inAltschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. Whenutilizing BLAST and Gapped BLAST programs, the default parameters of therespective programs (e.g., XBLAST and NBLAST) can be used.

In some instances, conservative mutations can be introduced into theCDRs in M0162-A04, e.g., at positions where the residues are not likelyto be involved in interacting with PKal as determined based on thecrystal structure. As used herein, a “conservative amino acidsubstitution” refers to an amino acid substitution that does not alterthe relative charge or size characteristics of the protein in which theamino acid substitution is made. Variants can be prepared according tomethods for altering polypeptide sequence known to one of ordinary skillin the art such as are found in references which compile such methods,e.g. Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds.,Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989, or Current Protocols in Molecular Biology, F. M. Ausubel, etal., eds., John Wiley & Sons, Inc., New York. Conservative substitutionsof amino acids include substitutions made amongst amino acids within thefollowing groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G;(e) S, T; (f) Q, N; and (g) E, D.

In some embodiments, the anti-PKal antibodies described here are notthose described in US 20110200611, which is incorporated by referenceherein.

In some embodiments, the anti-PKal antibodies described herein bind tothe same epitope as DX-2930 and/or compete for binding with DX-2930,with the proviso that the anti-PKal antibody is not DX-2930. In someembodiments, the anti-Pkal antibodies described herein bind to thesequence SWGE (SEQ ID NO: 48) and/or DACKG (SEQ ID NO: 49) in PKal. Insome embodiments, the anti-Pkal antibodies described herein do not bindto the sequence SWGE (SEQ ID NO: 48) and/or DACKG (SEQ ID NO: 49) inPkal. In some embodiments, the anti-Pkal antibodies described hereinbind to the sequence DGL, SEG, TSWGEG (SEQ ID NO: 50) and/or DACKG (SEQID NO: 49) in Pkal. In some embodiments, the anti-Pkal antibodiesdescribed herein do not bind to the sequence DGL, SEG, TSWGEG (SEQ IDNO: 50) and/or DACKG (SEQ ID NO: 49) in Pkal. In some embodiments, theanti-Pkal antibodies described herein do not bind to the sequence

(SEQ ID NO: 51) LVTNEECQKRYQDYKITQQ, (SEQ ID NO: 52) WVTGWGFSKEKGEI,(SEQ ID NO: 53) ACKGDSGGPL, (SEQ ID NO: 54) SWGDI, (SEQ ID NO: 55)HDIALIKL, (SEQ ID NO: 56) TPFSQIKEIIIHQNY, and/or (SEQ ID NO: 57)AHCFDGLPLQDVWRIY.

In some embodiments, the anti-PKal antibody described herein binds to anepitope located in the active domain of PKal (the whole epitope or aportion thereof) and is different from that DX-2930. The epitope of suchan antibody may have overlapping residues with those of the epitope ofDX-2930. Alternatively, there can be no overlapping residues between thetwo epitopes.

The sequences of the full length heavy chain and light chain of DX-2930are shown below.

DX-2930 Heavy Chain Amino Acid Sequence (451 amino acids)(SEQ ID NO: 46) EVQLLESGGGLVQPGGSLRLSCAASGFTFS HYIMM WVRQAPGKGLEWVSGIYSSGGITVYAD SVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAY RRIGVPRRDEFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSEELYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGDX-2930 Light Chain Amino Acid Sequence (213 amino acids)(SEQ ID NO: 47) DIQMTQSPSTLSASVGDRVTITC RASQSISSWLA WYQQKPGKAPKLLIYKASTLES GVPSRF SGSGSGTEFTLTISSLQPDDFATYYC QQYNTYWTFGQGTKVEIKRTVAAPSVFTFPPSDFQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

In the above sequences, the constant regions are italicized and the CDRregions are in boldface and underlined.

Antibody Preparation

Antibodies capable of binding PKal as described herein can be made byany method known in the art. See, for example, Harlow and Lane, (1988)Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, NewYork.

In some embodiments, antibodies specific to a target antigen (e.g., ahuman PKal or the catalytic domain thereof) can be made by theconventional hybridoma technology. The full-length target antigen or afragment thereof, optionally coupled to a carrier protein such as KLH,can be used to immunize a host animal for generating antibodies bindingto that antigen. The route and schedule of immunization of the hostanimal are generally in keeping with established and conventionaltechniques for antibody stimulation and production, as further describedherein. General techniques for production of mouse, humanized, and humanantibodies are known in the art and are described herein. It iscontemplated that any mammalian subject including humans or antibodyproducing cells therefrom can be manipulated to serve as the basis forproduction of mammalian, including human hybridoma cell lines.Typically, the host animal is inoculated intraperitoneally,intramuscularly, orally, subcutaneously, intraplantar, and/orintradermally with an amount of immunogen, including as describedherein.

Hybridomas can be prepared from the lymphocytes and immortalized myelomacells using the general somatic cell hybridization technique of Kohler,B. and Milstein, C. (1975) Nature 256:495-497 or as modified by Buck, D.W., et al., In Vitro, 18:377-381 (1982). Available myeloma lines,including but not limited to X63-Ag8.653 and those from the SalkInstitute, Cell Distribution Center, San Diego, Calif., USA, may be usedin the hybridization. Generally, the technique involves fusing myelomacells and lymphoid cells using a fusogen such as polyethylene glycol, orby electrical means well known to those skilled in the art. After thefusion, the cells are separated from the fusion medium and grown in aselective growth medium, such as hypoxanthine-aminopterin-thymidine(HAT) medium, to eliminate unhybridized parent cells. Any of the mediadescribed herein, supplemented with or without serum, can be used forculturing hybridomas that secrete monoclonal antibodies. As anotheralternative to the cell fusion technique, EBV immortalized B cells maybe used to produce the anti-PKal monoclonal antibodies described herein.The hybridomas are expanded and subcloned, if desired, and supernatantsare assayed for anti-immunogen activity by conventional immunoassayprocedures (e.g., radioimmunoassay, enzyme immunoassay, or fluorescenceimmunoassay).

Hybridomas that may be used as source of antibodies encompass allderivatives, progeny cells of the parent hybridomas that producemonoclonal antibodies capable of interfering with the PKal activity.Hybridomas that produce such antibodies may be grown in vitro or in vivousing known procedures. The monoclonal antibodies may be isolated fromthe culture media or body fluids, by conventional immunoglobulinpurification procedures such as ammonium sulfate precipitation, gelelectrophoresis, dialysis, chromatography, and ultrafiltration, ifdesired. Undesired activity if present, can be removed, for example, byrunning the preparation over adsorbents made of the immunogen attachedto a solid phase and eluting or releasing the desired antibodies off theimmunogen. Immunization of a host animal with a target antigen or afragment containing the target amino acid sequence conjugated to aprotein that is immunogenic in the species to be immunized, e.g.,keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, orsoybean trypsin inhibitor using a bifunctional or derivatizing agent,for example maleimidobenzoyl sulfosuccinimide ester (conjugation throughcysteine residues), N-hydroxysuccinimide (through lysine residues),glutaraldehyde, succinic anhydride, SOCl, or R1N═C═NR, where R and R1are different alkyl groups, can yield a population of antibodies (e.g.,monoclonal antibodies).

If desired, an antibody (monoclonal or polyclonal) of interest (e.g.,produced by a hybridoma) may be sequenced and the polynucleotidesequence may then be cloned into a vector for expression or propagation.The sequence encoding the antibody of interest may be maintained invector in a host cell and the host cell can then be expanded and frozenfor future use. In an alternative, the polynucleotide sequence may beused for genetic manipulation to “humanize” the antibody or to improvethe affinity (affinity maturation), or other characteristics of theantibody. For example, the constant region may be engineered to moreresemble human constant regions to avoid immune response if the antibodyis used in clinical trials and treatments in humans. It may be desirableto genetically manipulate the antibody sequence to obtain greateraffinity to the target antigen and greater efficacy in inhibiting theactivity of PKal. It will be apparent to one of skill in the art thatone or more polynucleotide changes can be made to the antibody and stillmaintain its binding specificity to the target antigen.

In other embodiments, fully human antibodies can be obtained by usingcommercially available mice that have been engineered to expressspecific human immunoglobulin proteins. Transgenic animals that aredesigned to produce a more desirable (e.g., fully human antibodies) ormore robust immune response may also be used for generation of humanizedor human antibodies. Examples of such technology are Xenomouse® fromAmgen, Inc. (Fremont, Calif.) and HuMAb-Mouse® and TC Mouse™ fromMedarex, Inc. (Princeton, N.J.). In another alternative, antibodies maybe made recombinantly by phage display or yeast technology. See, forexample, U.S. Pat. Nos. 5,565,332; 5,580,717; 5,733,743; and 6,265,150;and Winter et al., (1994) Annu. Rev. Immunol. 12:433-455, and.Alternatively, the phage display technology (McCafferty et al., (1990)Nature 348:552-553) can be used to produce human antibodies and antibodyfragments in vitro, from immunoglobulin variable (V) domain generepertoires from unimmunized donors.

Antigen-binding fragments of an intact antibody (full-length antibody)can be prepared via routine methods. For example, F(ab′)2 fragments canbe produced by pepsin digestion of an antibody molecule, and Fabfragments that can be generated by reducing the disulfide bridges ofF(ab′)2 fragments.

Genetically engineered antibodies, such as humanized antibodies,chimeric antibodies, single-chain antibodies, and bi-specificantibodies, can be produced via, e.g., conventional recombinanttechnology. In one example, DNA encoding a monoclonal antibodiesspecific to a target antigen can be readily isolated and sequenced usingconventional procedures (e.g., by using oligonucleotide probes that arecapable of binding specifically to genes encoding the heavy and lightchains of the monoclonal antibodies). The hybridoma cells serve as apreferred source of such DNA. Once isolated, the DNA may be placed intoone or more expression vectors, which are then transfected into hostcells such as E. coli cells, simian COS cells, Chinese hamster ovary(CHO) cells, or myeloma cells that do not otherwise produceimmunoglobulin protein, to obtain the synthesis of monoclonal antibodiesin the recombinant host cells. See, e.g., PCT Publication No. WO87/04462. The DNA can then be modified, for example, by substituting thecoding sequence for human heavy and light chain constant domains inplace of the homologous murine sequences, Morrison et al., (1984) Proc.Nat. Acad. Sci. 81:6851, or by covalently joining to the immunoglobulincoding sequence all or part of the coding sequence for anon-immunoglobulin polypeptide. In that manner, genetically engineeredantibodies, such as “chimeric” or “hybrid” antibodies; can be preparedthat have the binding specificity of a target antigen.

Techniques developed for the production of “chimeric antibodies” arewell known in the art. See, e.g., Morrison et al. (1984) Proc. Natl.Acad. Sci. USA 81, 6851; Neuberger et al. (1984) Nature 312, 604; andTakeda et al. (1984) Nature 314:452.

Methods for constructing humanized antibodies are also well known in theart. See, e.g., Queen et al., Proc. Natl. Acad. Sci. USA, 86:10029-10033(1989). In one example, variable regions of VH and VL of a parentnon-human antibody are subjected to three-dimensional molecular modelinganalysis following methods known in the art. Next, framework amino acidresidues predicted to be important for the formation of the correct CDRstructures are identified using the same molecular modeling analysis. Inparallel, human VH and VL chains having amino acid sequences that arehomologous to those of the parent non-human antibody are identified fromany antibody gene database using the parent VH and VL sequences assearch queries. Human VH and VL acceptor genes are then selected.

The CDR regions within the selected human acceptor genes can be replacedwith the CDR regions from the parent non-human antibody or functionalvariants thereof. When necessary, residues within the framework regionsof the parent chain that are predicted to be important in interactingwith the CDR regions (see above description) can be used to substitutefor the corresponding residues in the human acceptor genes.

A single-chain antibody can be prepared via recombinant technology bylinking a nucleotide sequence coding for a heavy chain variable regionand a nucleotide sequence coding for a light chain variable region.Preferably, a flexible linker is incorporated between the two variableregions. Alternatively, techniques described for the production ofsingle chain antibodies (U.S. Pat. Nos. 4,946,778 and 4,704,692) can beadapted to produce a phage or yeast scFv library and scFv clonesspecific to a PKal can be identified from the library following routineprocedures. Positive clones can be subjected to further screening toidentify those that inhibits PKal activity.

Antibodies obtained following a method known in the art and describedherein can be characterized using methods well known in the art. Forexample, one method is to identify the epitope to which the antigenbinds, or “epitope mapping.” There are many methods known in the art formapping and characterizing the location of epitopes on proteins,including solving the crystal structure of an antibody-antigen complex,competition assays, gene fragment expression assays, and syntheticpeptide-based assays, as described, for example, in Chapter 11 of Harlowand Lane, Using Antibodies, a Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1999. In an additionalexample, epitope mapping can be used to determine the sequence to whichan antibody binds. The epitope can be a linear epitope, i.e., containedin a single stretch of amino acids, or a conformational epitope formedby a three-dimensional interaction of amino acids that may notnecessarily be contained in a single stretch (primary structure linearsequence). Peptides of varying lengths (e.g., at least 4-6 amino acidslong) can be isolated or synthesized (e.g., recombinantly) and used forbinding assays with an antibody. In another example, the epitope towhich the antibody binds can be determined in a systematic screening byusing overlapping peptides derived from the target antigen sequence anddetermining binding by the antibody. According to the gene fragmentexpression assays, the open reading frame encoding the target antigen isfragmented either randomly or by specific genetic constructions and thereactivity of the expressed fragments of the antigen with the antibodyto be tested is determined. The gene fragments may, for example, beproduced by PCR and then transcribed and translated into protein invitro, in the presence of radioactive amino acids. The binding of theantibody to the radioactively labeled antigen fragments is thendetermined by immunoprecipitation and gel electrophoresis. Certainepitopes can also be identified by using large libraries of randompeptide sequences displayed on the surface of phage particles (phagelibraries). Alternatively, a defined library of overlapping peptidefragments can be tested for binding to the test antibody in simplebinding assays. In an additional example, mutagenesis of an antigenbinding domain, domain swapping experiments and alanine scanningmutagenesis can be performed to identify residues required, sufficient,and/or necessary for epitope binding. For example, domain swappingexperiments can be performed using a mutant of a target antigen in whichvarious fragments of the PKal polypeptide have been replaced (swapped)with sequences from a closely related, but antigenically distinctprotein (such as another member of the neurotrophin protein family). Byassessing binding of the antibody to the mutant PKal (e.g., thosemutants described in Example 2 below), the importance of the particularantigen fragment to antibody binding can be assessed.

Alternatively, competition assays can be performed using otherantibodies known to bind to the same antigen to determine whether anantibody binds to the same epitope as the other antibodies. Competitionassays are well known to those of skill in the art.

Any of the suitable methods known in the art, e.g., the epitope mappingmethods as described herein, can be applied to determine whether theanti-PKal antibody binds one or more of the specific residues/segmentsin the PKal as described herein. Further, the interaction of theantibody with one or more of those defined residues in PKal can bedetermined by routine technology. For example, a crystal structure canbe determined following the method disclosed in Example 1 below and thedistances between the residues in PKal and one or more residues in theantibody can be determined accordingly. Based on such distance, whethera specific residue in PKal interacts with one or more residues in theantibody can be determined. Further, suitable methods, such ascompetition assays and target mutagenesis assays can be applied todetermine the preferential binding of a candidate anti-PKal antibody tothe PKal as compared to another target such as a mutant PKal.

Pharmaceutical Compositions

One or more of the above-described anti-PKal antibodies can be mixedwith a pharmaceutically acceptable carrier (excipient), includingbuffer, to form a pharmaceutical composition for use in alleviating adisease or disorder that is associated with PKal. “Acceptable” meansthat the carrier must be compatible with the active ingredient of thecomposition (and preferably, capable of stabilizing the activeingredient) and not deleterious to the subject to be treated.Pharmaceutically acceptable excipients (carriers) including buffers,which are well known in the art. See, e.g., Remington: The Science andPractice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins,Ed. K. E. Hoover. In one example, a pharmaceutical composition describedherein contains more than one anti-PKal antibodies that recognizedifferent epitopes/residues of the target antigen.

The pharmaceutical compositions to be used in the present methods cancomprise pharmaceutically acceptable carriers, excipients, orstabilizers in the form of lyophilized formulations or aqueoussolutions. (Remington: The Science and Practice of Pharmacy 20th Ed.(2000) Lippincott Williams and Wilkins, Ed. K. E. Hoover). Acceptablecarriers, excipients, or stabilizers are nontoxic to recipients at thedosages and concentrations used, and may comprise buffers such asphosphate, citrate, and other organic acids; antioxidants includingascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrans; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).Pharmaceutically acceptable excipients are further described herein.

In some examples, the pharmaceutical composition described hereincomprises liposomes containing the anti-PKal antibody, which can beprepared by methods known in the art, such as described in Epstein, etal., Proc. Natl. Acad. Sci. USA 82:3688 (1985); Hwang, et al., Proc.Natl. Acad. Sci. USA 77:4030 (1980); and U.S. Pat. Nos. 4,485,045 and4,544,545. Liposomes with enhanced circulation time are disclosed inU.S. Pat. No. 5,013,556. Particularly useful liposomes can be generatedby the reverse phase evaporation method with a lipid compositioncomprising phosphatidylcholine, cholesterol and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter.

The anti-PKal antibody may also be entrapped in microcapsules prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are known in theart, see, e.g., Remington, The Science and Practice of Pharmacy 20th Ed.Mack Publishing (2000).

In other examples, the pharmaceutical composition described herein canbe formulated in sustained-release format. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g. films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(v nylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and 7ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), sucrose acetate isobutyrate, andpoly-D-(−)-3-hydroxybutyric acid.

The pharmaceutical compositions to be used for in vivo administrationmust be sterile. This is readily accomplished by, for example,filtration through sterile filtration membranes. Therapeutic antibodycompositions are generally placed into a container having a sterileaccess port, for example, an intravenous solution bag or vial having astopper pierceable by a hypodermic injection needle.

The pharmaceutical compositions described herein can be in unit dosageforms such as tablets, pills, capsules, powders, granules, solutions orsuspensions, or suppositories, for oral, parenteral or rectaladministration, or administration by inhalation or insufflation. Forpreparing solid compositions such as tablets, the principal activeingredient can be mixed with a pharmaceutical carrier, e.g. conventionaltableting ingredients such as corn starch, lactose, sucrose, sorbitol,talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, andother pharmaceutical diluents, e.g. water, to form a solidpreformulation composition containing a homogeneous mixture of acompound of the present invention, or a non-toxic pharmaceuticallyacceptable salt thereof. When referring to these preformulationcompositions as homogeneous, it is meant that the active ingredient isdispersed evenly throughout the composition so that the composition maybe readily subdivided into equally effective unit dosage forms such astablets, pills and capsules. This solid preformulation composition isthen subdivided into unit dosage forms of the type described abovecontaining from 0.1 to about 500 mg of the active ingredient of thepresent invention. The tablets or pills of the novel composition can becoated or otherwise compounded to provide a dosage form affording theadvantage of prolonged action. For example, the tablet or pill cancomprise an inner dosage and an outer dosage component, the latter beingin the form of an envelope over the former. The two components can beseparated by an enteric layer that serves to resist disintegration inthe stomach and permits the inner component to pass intact into theduodenum or to be delayed in release. A variety of materials can be usedfor such enteric layers or coatings, such materials including a numberof polymeric acids and mixtures of polymeric acids with such materialsas shellac, cetyl alcohol and cellulose acetate. Suitable surface-activeagents include, in particular, non-ionic agents, such aspolyoxyethylenesorbitans (e.g. Tween™ 20, 40, 60, 80 or 85) and othersorbitans (e.g. Span™ 20, 40, 60, 80 or 85). Compositions with asurface-active agent will conveniently comprise between 0.05 and 5%surface-active agent, and can be between 0.1 and 2.5%. It will beappreciated that other ingredients may be added, for example mannitol orother pharmaceutically acceptable vehicles, if necessary.

Suitable emulsions may be prepared using commercially available fatemulsions, such as Intralipid™, Liposyn™, Infonutrol™, Lipofundin™ andLipiphysan™. The active ingredient may be either dissolved in apre-mixed emulsion composition or alternatively it may be dissolved inan oil (e.g. soybean oil, safflower oil, cottonseed oil, sesame oil,corn oil or almond oil) and an emulsion formed upon mixing with aphospholipid (e.g. egg phospholipids, soybean phospholipids or soybeanlecithin) and water. It will be appreciated that other ingredients maybe added, for example glycerol or glucose, to adjust the tonicity of theemulsion. Suitable emulsions will typically contain up to 20% oil, forexample, between 5 and 20%. The fat emulsion can comprise fat dropletsbetween 0.1 and 1.0.im, particularly 0.1 and 0.5.im, and have a pH inthe range of 5.5 to 8.0.

The emulsion compositions can be those prepared by mixing an anti-PKalantibody with Intralipid™ or the components thereof (soybean oil, eggphospholipids, glycerol and water).

Pharmaceutical compositions for inhalation or insufflation includesolutions and suspensions in pharmaceutically acceptable, aqueous ororganic solvents, or mixtures thereof, and powders. The liquid or solidcompositions may contain suitable pharmaceutically acceptable excipientsas set out above. In some embodiments, the compositions are administeredby the oral or nasal respiratory route for local or systemic effect.

Compositions in preferably sterile pharmaceutically acceptable solventsmay be nebulised by use of gases. Nebulised solutions may be breatheddirectly from the nebulising device or the nebulising device may beattached to a face mask, tent or intermittent positive pressurebreathing machine. Solution, suspension or powder compositions may beadministered, preferably orally or nasally, from devices which deliverthe formulation in an appropriate manner.

Use of anti-PKal Antibodies for Treating Diseases/Disorders Associatedwith Plasma Kallikrein

The anti-PKal antibodies described herein would be effective in treatinga disease or disorder associated the PKal. Examples of such diseases andconditions which can be treated (or prevented) by a plasma kallikreinbinding protein described herein include: rheumatoid arthritis, gout,intestinal bowel disease, oral mucositis, neuropathic pain, inflammatorypain, spinal stenosis-degenerative spine disease, arterial or venousthrombosis, post operative ileus, aortic aneurysm, osteoarthritis,vasculitis, edema, hereditary angioedema, cerebral edema, pulmonaryembolism, stroke, clotting induced by ventricular assistance devices orstents, head trauma or peri-tumor brain edema, sepsis, acute middlecerebral artery (MCA) ischemic event (stroke), restenosis (e.g., afterangioplasty), systemic lupus erythematosis nephritis, burn injury, andDME. A plasma kallikrein binding protein described herein can also beused to promote wound healing. A plasma kallikrein binding proteindescribed herein can also be used as an oncology treatment by mechanismsthat include, but are not limited to, blocking production ofpro-angiogenic bradykinin.

To practice the method disclosed herein, an effective amount of thepharmaceutical composition described above can be administered to asubject (e.g., a human) in need of the treatment via a suitable route,such as intravenous administration, e.g., as a bolus or by continuousinfusion over a period of time, by intramuscular, intraperitoneal,intracerebrospinal, subcutaneous, intra-articular, intrasynovial,intrathecal, oral, inhalation or topical routes. Commercially availablenebulizers for liquid formulations, including jet nebulizers andultrasonic nebulizers are useful for administration. Liquid formulationscan be directly nebulized and lyophilized powder can be nebulized afterreconstitution. Alternatively, anti-PKal antibodies can be aerosolizedusing a fluorocarbon formulation and a metered dose inhaler, or inhaledas a lyophilized and milled powder.

The subject to be treated by the methods described herein can be amammal, more preferably a human. Mammals include, but are not limitedto, farm animals, sport animals, pets, primates, horses, dogs, cats,mice and rats. A human subject who needs the treatment may be a humanpatient having, at risk for, or suspected of having a disease/disorderassociated with PKal, such as those noted above. A subject having aPKal-associated disease or disorder can be identified by routine medicalexamination, e.g., laboratory tests, organ functional tests, CT scans,or ultrasounds. A subject suspected of having any of suchdisease/disorder might show one or more symptoms of thedisease/disorder. A subject at risk for the disease/disorder can be asubject having one or more of the risk factors for thatdisease/disorder.

“An effective amount” as used herein refers to the amount of each activeagent required to confer therapeutic effect on the subject, either aloneor in combination with one or more other active agents. Effectiveamounts vary, as recognized by those skilled in the art, depending onthe particular condition being treated, the severity of the condition,the individual patient parameters including age, physical condition,size, gender and weight, the duration of the treatment, the nature ofconcurrent therapy (if any), the specific route of administration andlike factors within the knowledge and expertise of the healthpractitioner. These factors are well known to those of ordinary skill inthe art and can be addressed with no more than routine experimentation.It is generally preferred that a maximum dose of the individualcomponents or combinations thereof be used, that is, the highest safedose according to sound medical judgment. It will be understood by thoseof ordinary skill in the art, however, that a patient may insist upon alower dose or tolerable dose for medical reasons, psychological reasonsor for virtually any other reasons.

Empirical considerations, such as the half-life, generally willcontribute to the determination of the dosage. For example, antibodiesthat are compatible with the human immune system, such as humanizedantibodies or fully human antibodies, may be used to prolong half-lifeof the antibody and to prevent the antibody being attacked by the host'simmune system. Frequency of administration may be determined andadjusted over the course of therapy, and is generally, but notnecessarily, based on treatment and/or suppression and/or ameliorationand/or delay of a disease/disorder associated with PKal. Alternatively,sustained continuous release formulations of an anti-PKal may beappropriate. Various formulations and devices for achieving sustainedrelease are known in the art.

In one example, dosages for an anti-PKal antibody as described hereinmay be determined empirically in individuals who have been given one ormore administration(s) of the antibody. Individuals are givenincremental dosages of the antagonist. To assess efficacy of theantagonist, an indicator of the disease/disorder can be followed.

Generally, for administration of any of the antibodies described herein,an initial candidate dosage can be about 2 mg/kg. For the purpose of thepresent disclosure, a typical daily dosage might range from about any of0.1 μg/kg to 3 μg/kg to 30 μg/kg to 300 μg/kg to 3 mg/kg, to 30 mg/kg to100 mg/kg or more, depending on the factors mentioned above. Forrepeated administrations over several days or longer, depending on thecondition, the treatment is sustained until a desired suppression ofsymptoms occurs or until sufficient therapeutic levels are achieved toalleviate a disease or disorder associated with PKal, or a symptomthereof. An exemplary dosing regimen comprises administering an initialdose of about 2 mg/kg, followed by a weekly maintenance dose of about 1mg/kg of the antibody, or followed by a maintenance dose of about 1mg/kg every other week. However, other dosage regimens may be useful,depending on the pattern of pharmacokinetic decay that the practitionerwishes to achieve. For example, dosing from one-four times a week iscontemplated. In some embodiments, dosing ranging from about 3 μg/mg toabout 2 mg/kg (such as about 3 μg/mg, about 10 μg/mg, about 30 μg/mg,about 100 μg/mg, about 300 μg/mg, about 1 mg/kg, and about 2 mg/kg) maybe used. In some embodiments, dosing frequency is once every week, every2 weeks, every 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks,every 8 weeks, every 9 weeks, or every 10 weeks; or once every month,every 2 months, or every 3 months, or longer. The progress of thistherapy is easily monitored by conventional techniques and assays. Thedosing regimen (including the antibody used) can vary over time.

In some embodiments, for an adult patient of normal weight, dosesranging from about 0.3 to 5.00 mg/kg may be administered. The particulardosage regimen, i.e., dose, timing and repetition, will depend on theparticular individual and that individual's medical history, as well asthe properties of the individual agents (such as the half-life of theagent, and other considerations well known in the art).

For the purpose of the present disclosure, the appropriate dosage of ananti-PKal antibody will depend on the specific antibody (or compositionsthereof) employed, the type and severity of the disease/disorder,whether the antibody is administered for preventive or therapeuticpurposes, previous therapy, the patient's clinical history and responseto the antagonist, and the discretion of the attending physician.Typically the clinician will administer an anti-PKal antibody, until adosage is reached that achieves the desired result. Administration of ananti-PKal antibody can be continuous or intermittent, depending, forexample, upon the recipient's physiological condition, whether thepurpose of the administration is therapeutic or prophylactic, and otherfactors known to skilled practitioners. The administration of ananti-PKal antibody may be essentially continuous over a preselectedperiod of time or may be in a series of spaced dose, e.g., eitherbefore, during, or after developing a disease or disorder associatedwith PKal.

As used herein, the term “treating” refers to the application oradministration of a composition including one or more active agents to asubject, who has a disease/disorder associated with PKal, a symptom ofthe disease/disorder, or a predisposition toward the disease/disorder,with the purpose to cure, heal, alleviate, relieve, alter, remedy,ameliorate, improve, or affect the disorder, the symptom of the disease,or the predisposition toward the disease/disorder.

Alleviating a disease/disorder associated with PKal includes delayingthe development or progression of the disease, or reducing diseaseseverity. Alleviating the disease does not necessarily require curativeresults. As used therein, “delaying” the development of adisease/disorder associated with PKal means to defer, hinder, slow,retard, stabilize, and/or postpone progression of the disease. Thisdelay can be of varying lengths of time, depending on the history of thedisease and/or individuals being treated. A method that “delays” oralleviates the development of a disease, or delays the onset of thedisease, is a method that reduces probability of developing one or moresymptoms of the disease in a given time frame and/or reduces extent ofthe symptoms in a given time frame, when compared to not using themethod. Such comparisons are typically based on clinical studies, usinga number of subjects sufficient to give a statistically significantresult.

“Development” or “progression” of a disease means initial manifestationsand/or ensuing progression of the disease. Development of the diseasecan be detectable and assessed using standard clinical techniques aswell known in the art. However, development also refers to progressionthat may be undetectable. For purpose of this disclosure, development orprogression refers to the biological course of the symptoms.“Development” includes occurrence, recurrence, and onset. As used herein“onset” or “occurrence” of a disease/disorder associated with PKalincludes initial onset and/or recurrence.

In some embodiments, the anti-PKal antibody described herein isadministered to a subject in need of the treatment at an amountsufficient to inhibit the activity of PKal by at least 20% (e.g., 30%,40%, 50%, 60%, 70%, 80%, 90% or greater) in vivo. In other embodiments,the antibody is administered in an amount effective in reducing the PKallevel by at least 20% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90% orgreater).

Conventional methods, known to those of ordinary skill in the art ofmedicine, can be used to administer the pharmaceutical composition tothe subject, depending upon the type of disease to be treated or thesite of the disease. This composition can also be administered via otherconventional routes, e.g., administered orally, parenterally, byinhalation spray, topically, rectally, nasally, buccally, vaginally orvia an implanted reservoir. The term “parenteral” as used hereinincludes subcutaneous, intracutaneous, intravenous, intramuscular,intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal,intralesional, and intracranial injection or infusion techniques. Inaddition, it can be administered to the subject via injectable depotroutes of administration such as using 1-, 3-, or 6-month depotinjectable or biodegradable materials and methods.

Injectable compositions may contain various carriers such as vegetableoils, dimethylactamide, dimethyformamide, ethyl lactate, ethylcarbonate, isopropyl myristate, ethanol, and polyols (glycerol,propylene glycol, liquid polyethylene glycol, and the like). Forintravenous injection, water soluble antibodies can be administered bythe drip method, whereby a pharmaceutical formulation containing theantibody and a physiologically acceptable excipients is infused.Physiologically acceptable excipients may include, for example, 5%dextrose, 0.9% saline, Ringer's solution or other suitable excipients.Intramuscular preparations, e.g., a sterile formulation of a suitablesoluble salt form of the antibody, can be dissolved and administered ina pharmaceutical excipient such as Water-for-Injection, 0.9% saline, or5% glucose solution.

In one embodiment, an anti-PKal antibody is administered viasite-specific or targeted local delivery techniques. Examples ofsite-specific or targeted local delivery techniques include variousimplantable depot sources of the anti-PKal antibody or local deliverycatheters, such as infusion catheters, an indwelling catheter, or aneedle catheter, synthetic grafts, adventitial wraps, shunts and stentsor other implantable devices, site specific carriers, direct injection,or direct application. See, e.g., PCT Publication No. WO 00/53211 andU.S. Pat. No. 5,981,568.

Targeted delivery of therapeutic compositions containing an antisensepolynucleotide, expression vector, or subgenomic polynucleotides canalso be used. Receptor-mediated DNA delivery techniques are describedin, for example, Findeis et al., Trends Biotechnol. (1993) 11:202; Chiouet al., Gene Therapeutics: Methods And Applications Of Direct GeneTransfer (J. A. Wolff, ed.) (1994); Wu et al., J. Biol. Chem. (1988)263:621; Wu et al., J. Biol. Chem. (1994) 269:542; Zenke et al., Proc.Natl. Acad. Sci. USA (1990) 87:3655; Wu et al., J. Biol. Chem. (1991)266:338.

Therapeutic compositions containing a polynucleotide (e.g., thoseencoding the anti-PKal antibodies described herein) are administered ina range of about 100 ng to about 200 mg of DNA for local administrationin a gene therapy protocol. In some embodiments, concentration ranges ofabout 500 ng to about 50 mg, about 1 μg to about 2 mg, about 5 μg toabout 500 μg, and about 20 μg to about 100 μg of DNA or more can also beused during a gene therapy protocol.

The therapeutic polynucleotides and polypeptides described herein can bedelivered using gene delivery vehicles. The gene delivery vehicle can beof viral or non-viral origin (see generally, Jolly, Cancer Gene Therapy(1994) 1:51; Kimura, Human Gene Therapy (1994) 5:845; Connelly, HumanGene Therapy (1995) 1:185; and Kaplitt, Nature Genetics (1994) 6:148).Expression of such coding sequences can be induced using endogenousmammalian or heterologous promoters and/or enhancers. Expression of thecoding sequence can be either constitutive or regulated.

Viral-based vectors for delivery of a desired polynucleotide andexpression in a desired cell are well known in the art. Exemplaryviral-based vehicles include, but are not limited to, recombinantretroviruses (see, e.g., PCT Publication Nos. WO 90/07936; WO 94/03622;WO 93/25698; WO 93/25234; WO 93/11230; WO 93/10218; WO 91/02805; U.S.Pat. Nos. 5,219,740 and 4,777,127; GB Patent No. 2,200,651; and EPPatent No. 0 345 242), alphavirus-based vectors (e.g., Sindbis virusvectors, Semliki forest virus (ATCC VR-67; ATCC VR-1247), Ross Rivervirus (ATCC VR-373; ATCC VR-1246) and Venezuelan equine encephalitisvirus (ATCC VR-923; ATCC VR-1250; ATCC VR 1249; ATCC VR-532)), andadeno-associated virus (AAV) vectors (see, e.g., PCT Publication Nos. WO94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO95/00655). Administration of DNA linked to killed adenovirus asdescribed in Curiel, Hum. Gene Ther. (1992) 3:147 can also be employed.

Non-viral delivery vehicles and methods can also be employed, including,but not limited to, polycationic condensed DNA linked or unlinked tokilled adenovirus alone (see, e.g., Curiel, Hum. Gene Ther. (1992)3:147); ligand-linked DNA (see, e.g., Wu, J. Biol. Chem. (1989)264:16985); eukaryotic cell delivery vehicles cells (see, e.g., U.S.Pat. No. 5,814,482; PCT Publication Nos. WO 95/07994; WO 96/17072; WO95/30763; and WO 97/42338) and nucleic charge neutralization or fusionwith cell membranes. Naked DNA can also be employed. Exemplary naked DNAintroduction methods are described in PCT Publication No. WO 90/11092and U.S. Pat. No. 5,580,859. Liposomes that can act as gene deliveryvehicles are described in U.S. Pat. No. 5,422,120; PCT Publication Nos.WO 95/13796; WO 94/23697; WO 91/14445; and EP Patent No. 0524968.Additional approaches are described in Philip, Mol. Cell. Biol. (1994)14:2411, and in Woffendin, Proc. Natl. Acad. Sci. (1994) 91:1581.

The particular dosage regimen, i.e., dose, timing and repetition, usedin the method described herein will depend on the particular subject andthat subject's medical history. In some embodiments, more than oneanti-PKal antibodies, or a combination of an anti-PKal antibody andanother suitable therapeutic agent, may be administered to a subject inneed of the treatment. The antagonist can be the same type or differentfrom each other. The anti-PKal antibody can also be used in conjunctionwith other agents that serve to enhance and/or complement theeffectiveness of the agents.

Treatment efficacy for a disease/disorder associated with PKal can beassessed by methods well-known in the art.

Kits for Use in Alleviating Diseases/Disorders Associated with PlasmaKallikrein

The present disclosure also provides kits for use in alleviatingdiseases/disorders associated with plasma kallikrein. Such kits caninclude one or more containers comprising an anti-PKal antibody, e.g.,any of those described herein.

In some embodiments, the kit can comprise instructions for use inaccordance with any of the methods described herein. The includedinstructions can comprise a description of administration of theanti-PKal antibody to treat, delay the onset, or alleviate a targetdisease as those described herein. The kit may further comprise adescription of selecting an individual suitable for treatment based onidentifying whether that individual has the target disease. In stillother embodiments, the instructions comprise a description ofadministering an antibody to an individual at risk of the targetdisease.

The instructions relating to the use of an anti-PKal antibody generallyinclude information as to dosage, dosing schedule, and route ofadministration for the intended treatment. The containers may be unitdoses, bulk packages (e.g., multi-dose packages) or sub-unit doses.Instructions supplied in the kits of the invention are typically writteninstructions on a label or package insert (e.g., a paper sheet includedin the kit), but machine-readable instructions (e.g., instructionscarried on a magnetic or optical storage disk) are also acceptable.

The label or package insert indicates that the composition is used fortreating, delaying the onset and/or alleviating a disease or disorderassociated with PKal. Instructions may be provided for practicing any ofthe methods described herein.

The kits of this invention are in suitable packaging. Suitable packagingincludes, but is not limited to, vials, bottles, jars, flexiblepackaging (e.g., sealed Mylar or plastic bags), and the like. Alsocontemplated are packages for use in combination with a specific device,such as an inhaler, nasal administration device (e.g., an atomizer) oran infusion device such as a minipump. A kit may have a sterile accessport (for example the container may be an intravenous solution bag or avial having a stopper pierceable by a hypodermic injection needle). Thecontainer may also have a sterile access port (for example the containermay be an intravenous solution bag or a vial having a stopper pierceableby a hypodermic injection needle). At least one active agent in thecomposition is an anti-PKal antibody as those described herein.

Kits may optionally provide additional components such as buffers andinterpretive information. Normally, the kit comprises a container and alabel or package insert(s) on or associated with the container. In someembodiments, the invention provides articles of manufacture comprisingcontents of the kits described above.

General Techniques

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry andimmunology, which are within the skill of the art. Such techniques areexplained fully in the literature, such as, Molecular Cloning: ALaboratory Manual, second edition (Sambrook, et al., 1989) Cold SpringHarbor Press; Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Methodsin Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook(J. E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R. I.Freshney, ed., 1987); Introduction to Cell and Tissue Culture (J. P.Mather and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture:Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell,eds., 1993-8) J. Wiley and Sons; Methods in Enzymology (Academic Press,Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C.Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J. M.Miller and M. P. Calos, eds., 1987); Current Protocols in MolecularBiology (F. M. Ausubel, et al., eds., 1987); PCR: The Polymerase ChainReaction, (Mullis, et al., eds., 1994); Current Protocols in Immunology(J. E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology(Wiley and Sons, 1999); Immunobiology (C. A. Janeway and P. Travers,1997); Antibodies (P. Finch, 1997); Antibodies: a practical approach (D.Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a practicalapproach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000);Using antibodies: a laboratory manual (E. Harlow and D. Lane (ColdSpring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J.D. Capra, eds., Harwood Academic Publishers, 1995).

Without further elaboration, it is believed that one skilled in the artcan, based on the above description, utilize the present invention toits fullest extent. The following specific embodiments are, therefore,to be construed as merely illustrative, and not limitative of theremainder of the disclosure in any way whatsoever. All publicationscited herein are incorporated by reference for the purposes or subjectmatter referenced herein.

Example 1: Identification of Critical Residues in the Catalytic Domainof Human Plasma Kallikrein Based on Crystal Structures of DX-2930-PKalComplex

The catalytic domain of human plasma kallikrein (FIG. 2 ), fused with aHis-tag, was expressed in insect cells and purified initially by anickel affinity column. The His-tag was removed from the plasmakallikrein via trypsin digestion and the free plasma kallikrein waspurified by a benzamidine affinity column, followed by a SEC column. Thepurified product was examined on a PAGE gel. The result indicates thatthe catalytic domain of human plasma kallikrein was properly expressedand purified.

DX-2930 was prepared via routine recombinant technology and purified. Arecombinant Fab fragment of DX-2930 was produced via routine method andpurified.

The DX-2930 Fab fragment and the catalytic domain of human plasmakallikrein were mixed at various concentrations under suitableconditions allowing formation of antibody-PKal complexes. The complexesthus formed were examined using HPLC to determine the antibody-PKalratio in the complexes. Accordingly, the suitable concentrations of boththe antibody and the PKal were identified for formation of a 1:1complex.

The antibody-PKal complex was kept under various conditions allowing forcrystallization. Diffraction analysis was performed on the crystallizedcomplex. The crystal structures (2.1 Å and 2.4 Å) were determined basedon the diffraction statistics.

According to the crystal structures, residues in the catalytic domain ofhuman Pkal that are involved in the interaction with DX-2930 wereidentified. These residues are indicated (boldfaced and underlined) inFIG. 2 , which provides the amino acid sequence of the catalytic domainof human PKal (residues 391-638 of human PKal).

In addition, residues in DX-2930 that interact with PKal were alsoidentified based on the crystal structure, including E1, V2, F27, T28,F29, S30, H31, R100, 1101, G102, V103, P104, R105, R106, D107, G107,K108, and D111 in the heavy chain variable region, and S31, W32, Y49,K50, T53, L54, E55, 556, G57, and V58 in the light chain variableregion.

These results indicate that HC CDR3 of DX-2930 is the main region thatinteracts with PKal (see FIG. 1 ) and a couple of residues in the HCCDR1 and FR1 might also contribute to the interaction with PKal. In thelight chain, the LC CDR2 region was found to contribute to theinteraction.

Further, the results also indicate that variations at certain positionswith the HC CDR3 region may be allowed. For example, position 103requires small hydrophobic residues such as V or I. As another example,R106 may be replaced with W, and E108 may be replaced with S or Dwithout substantially affecting the PKal binding activity. Similarly,D110 might be replaced with E.

Example 2: Affinity Maturation Results Match Structural InformationDerived from Crystal Structure

The heavy chain variable region, particularly the HC CDR3 region, ofantibody M0162-A04 was subject to affinity maturation. Various mutantshaving amino acid variations at one or more positions in the HC CDR3region were generated and their K_(i,app) values were determinedfollowing routine methods.

Briefly, PKal and a Fab at various concentrations are incubated togetherfor 1 hour at 30° C. A substrate peptide (cleavable by PKal) is thenadded to this PKal-Fab mixture. The rate of substrate peptidecleavage/proteolysis is then measured, and plotted against theconcentrations of the Fab. This plot is then fit to the Morrisonequation, which calculates the K_(i,app) value. The results thusobtained are shown in FIG. 3 and Table 2 below:

TABLE 2 Summary of Hv-CDR3 Affinity Maturation Results Ki, appInitial Name Hv CDR3 (nM) SEQ ID NO M0162-A04 RRTGIPRRDAFDI    2.5  1M0199-A11 --R----------    2  2 M0201-F11 --S----------    3  3M0202-A08 -------W-----    2.8  4 M0201-A06 ---------V---    3.8  5M0202-E03 -----------E-    2  6 M0199-B01 ------------N    1.6  7M0200-B01 ------------S    3.6  8 M0201-H06 ----V--------    0.6  9M0202-H05 ----V----V---    0.26 10 M0201-H08 ----V-----L-N    0.8 11M0200-E11 ----V-------N    0.4 12 M0200-H07 ----V---N---N    0.4 13M0202-F06 ----V--W-----    0.33 14 M0200-A10 ----V----S---     0.25 15M0202-G03 ----V----S-E-    0.4 16 M0202-A12 Q---V----S-N-    0.1 17M0202-H03 ----V--W-D---    0.1 18 M0201-A07 ----V----E---    0.1 19M0202-C02 --P-V--------    0.6 20 M0202-B04 --S-V--------    0.2 21M0202-E06 --R-V----D---    0.06 22 M0202-A01 --I-V--------    0.3 23M0202-D09 --I-V----S---     0.2 24 M0200-D03 --I-V----S--M    0.1 25M0202-C09 --I-V----D---    0.06 26 M0199-A08 --I-V----E---    0.06 27X133-B02 --I----------    2.2 28 X133-D06 --I------E---    0.33 29X135-A01 ----A--------  247.7 30 X133-G05 ----S-------- 1405.6 31X133-F10 ----L--------   14.7 32 X135-A03 ---------E---    1.1 33

The affinity maturation results indicate that variations at certainpositions within the HC CDR3 region result in high affinity/inhibitoryanti-PKal antibodies as compared to the parent M0162-A04 clone. Theseresults match with the structural information provided in Example 1above. Note that the HC CDR3 region of clone M0199-AOS is identical tothat of DX-2930.

Example 3: Impact of Mutations in Plasma Kallikrein on AntibodyInhibitory Activity

The inhibitory activities of mutant X115-F02 against various PKalmutants were examined.

X115-F02 is an JgG that is the same as DX-2930 except that it contains aC-terminal lysine residue not present in DX-2930 and was expressed inHEK293T cells rather than CHO cells (Table 1 above). The bindingspecificity and affinity of X115-F02 is the same as DX-2930.

The wild type and four mutants of plasma kallikrein used in this study(FIG. 5 ) are recombinant catalytic domains expressed and purified fromPichia pastoris. Mutant 1 contains the following mutations in the S3subsite of the active site: S478A, N481A, S506A, Y507A) (numbers basedon the full length prekallikrein amino acid sequence). Mutant 2 containsthe following mutations in the S1′ subsite of the active site: R551A,Q553A, Y555A, T558A, R560A. Mutant 4 contains the following mutationsthat are distal from the active site: N396A, S398A, W399A. Mutant 3 wasfound to be inactive and therefore was not tested in the activity assay.Mutant 3 contains the following mutations in the S1′ subsite of theactive site: D572A, K575A, D577A.

The inhibitory activity of X115-F02 against the wild-type PKal and themutants were carried out using the method described in Example 2 aboveand the K_(i,app) values were determined. As shown in FIG. 4 , themutations in Mutant 1 and 4 did not significantly affect the potency ofX115-F02 inhibition of plasma kallikrein. Surprisingly, the mutations inMutant 2 reduced the potency approximately 65-fold. These resultsindicate that residues R551A, Q553A, Y555A, T558A, R560A and theiradjacent residues might be important to the inhibitory activity ofX115-F02 (DX-2930).

Other Embodiments

All of the features disclosed in this specification may be combined inany combination. Each feature disclosed in this specification may bereplaced by an alternative feature serving the same, equivalent, orsimilar purpose. Thus, unless expressly stated otherwise, each featuredisclosed is only an example of a generic series of equivalent orsimilar features.

From the above description, one skilled in the art can easily ascertainthe essential characteristics of the present invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions. Thus, other embodiments are also within the claims.

1.-31. (canceled)
 32. A method for inhibiting plasma kallikrein (pKal)activity, comprising administering to a subject an effective amount ofan antibody that binds pKal, wherein the antibody comprises: a heavychain variable region that comprises a complementarity determiningregion 1 (HC CDR1) comprising the sequence HYIMM, a complementaritydetermining region 2 (HC CDR2) comprising the sequenceGIYSSGGITVYADSVKG, and a complementarity determining region 3 (HC CDR3)comprising the sequence (HC CDR3) comprising the sequence (i)(SEQ ID NO: 28) RRIGIPRRDAFDI; (ii) (SEQ ID NO: 29) RRIGIPRRDEFDI; (iii)(SEQ ID NO: 30) RRTGAPRRDAFDI; (iv)  (SEQ ID NO: 31) RRTGSPRRDAFDI; (v)(SEQ ID NO: 32) RRTGLPRRDAFDI; or (vi) (SEQ ID NO: 33) RRTGIPRRDEFDI;

a light chain (LC) variable region that comprises a complementaritydetermining region 1 (LC CDR1) comprising the sequence RASQSISSWLA, acomplementarity determining region 2 (LC CDR2) comprising the sequenceKASTLES, and a complementarity determining region 3 (LC CDR3) comprisingthe sequence QQYNTYWT.
 33. The method of claim 32, wherein the antibodyis a full-length antibody or an antigen-binding fragment thereof. 34.The method of claim 33, wherein the antibody is an IgG.
 35. The methodof claim 32, wherein the antibody is a human antibody or a humanizedantibody.
 36. The method of claim 32, wherein the antibody inhibits theactivity of pKal by at least 80%.
 37. The method of claim 32, whereinthe antibody has a Ki_(,app) lower than about 1 nM.
 38. The method ofclaim 32, wherein the light chain variable region further includes G₅₇in the framework region 3 (FR3).
 39. The method of claim 32, wherein thelight chain variable region further includes N₄₅ in the framework region2 (FR2).
 40. The method of claim 32, wherein the antibody preferentiallybinds an active pKal.
 41. The method of claim 32, wherein the subject isa human patient diagnosed with, suspected of having, or at risk for adisease.
 42. The method of claim 41, wherein the disease is rheumatoidarthritis, gout, intestinal bowel disease, oral mucositis, neuropathicpain, inflammatory pain, spinal stenosis-degenerative spine disease,arterial or venous thrombosis, post-operative ileus, aortic aneurysm,osteoarthritis, vasculitis, edema, hereditary angioedema, cerebraledema, pulmonary embolism, stroke, clotting induced by ventricularassistance devices or stents, head trauma or peri-tumor brain edema,sepsis, acute middle cerebral artery (MCA) ischemic event (stroke),restenosis (e.g., after angioplasty), systemic lupus erythematosisnephritis, burn injury, or diabetic macular edema.